Wide-field microscopy provides images with short acquisition time and a wide field of view, however its inability to resolve depth information limits it to near-surface measurements or thin samples. In order to provide both lateral and axial information, many techniques in microscopy have been explored. In particular, confocal microscopy has gained popularity for its ability to section individual planes by passing the light through a pinhole and eliminating out of focus light. In particular, confocal reflectance microscopy (CRM) has been used for imaging skin. However, CRM requires the ability to scan individual points on an object leading to expensive and elaborate point scanning equipment. Other techniques for scanning a full 2D plane include the use of dual-wedge scanners or 2D micro-electrical-mechanical scanners.
Apart from microscopy techniques that require lateral scanning, other optical microscopy techniques have been introduced that capture an entire 2D image field in a single step. One such technique is known as structured illumination. This technique uses a known pattern, typically at a constant spatial frequency, projected onto a sample. Reflected light from areas conjugate to the pattern is modulated at that spatial frequency and can be separated from the out of focus regions. This technique requires that the spatial frequency of the pattern is known a-priori, such that an exact ⅓ phase shift can be applied to resolve the entire image. An extension of this technique, known as dynamic speckle illumination (DSI) uses a coherent laser source and a randomly distributed speckle pattern produced by a spatial light modulator that is decorrelated from image to image by either translation or randomization. This technique has been successfully utilized for optical microscopy, however the number of images required to produce sectioning increases with depth, reducing the quality of the sectioning when imaging deeper into the specimen. Also, areas where speckles are correlated can result in streaking within the image, causing undesired artifacts within the final image. Dynamic speckle illumination has also typically been utilized with fluorescence from a specimen as a means of providing depth information about the specimen. The use of fluorescence requires that light be absorped and re-emitted by the specimen being imaged, which often requires the staining of a specimen with a fluorescent material. Fluorescent stains may suffer from limitations in the potential wavelengths that can be emitted, are subject to photobleaching, and can potentially be harmful to a specimen being imaged in-vivo.
Accordingly, there is a need for optical microscopy systems and techniques that provide for rapid, full-field image acquisition, high quality image sectioning at depth, and safety for in-vivo measurements, all while being very cost effective to produce. Such systems and techniques will increase the range of environments and applications where optical microscopy can be utilized. When applied to biomedical and healthcare applications, such systems and techniques can shorten diagnostic times, reduce invasiveness of imaging procedures, and improve the quality of patient care.